Shiga toxin (Stx) producing E. coli (STEC) are foodborne pathogens that can cause severe morbidity and mortality, including hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). They are classified as category B bioterrorism select agents. A recent outbreak of Shiga toxin 2 (Stx2) producing E. coli in Germany represented one of the largest outbreaks of HUS worldwide and was the deadliest on record. HUS is the most common cause of renal failure in infants and young children in the US. There are no specific protective measures or therapeutics effective against infection by bacteria producing these toxins. Shiga toxins are a family of AB5 toxins or type II ribosome-inactivating proteins (RIPs), consisting of an enzymatically active A subunit that associates with a pentamer of identical B subunits. The A subunit is an N-glycosidase that specifically removes an adenine from the -sarcin/ricin loop (SRL) of 28S rRNA, resulting in inhibition of protein synthesis. E. coli strains producing Stx2 are more likely to be associated with progression to HUS than strains producing Stx1. The mechanism that accounts for the differences in cytotoxicity of Stx1 and Stx2 is not known. We have developed the yeast, Saccharomyces cerevisae, as a powerful model to study the cytotoxicity of RIPs. Using this model, we showed that ribosomal stalk is critical for Stx1A and Stx2A to depurinate the SRL. Our preliminary data indicates that Stx1A and Stx2A respond differently to mutations in the ribosomal stalk. We show for the first time that Stx2 has higher affinity for the stalk than Stx1. We propose to examine the interaction of Stx1 and Stx2 with ribosomes from yeast and mammalian cells to test the hypothesis that they differ in their requirements for the ribosomal stalk and ultimately their interaction with the stalk is critical for ribosome depurination and cytotoxicity. We will determine if ribosome specificity of Shiga toxins is due to their interactions with the stalk and if peptides corresponding to the recognition sequences of Stx2A1 can block toxin activity. Identifying the mechanistic differences in binding of Shiga toxins to ribosomes would provide a major step towards understanding how these toxins work and how to block their activity. Since the A and the B subunits of Stx2 responsible for the German outbreak are identical in amino acid sequence to the Stx2 we are studying in our lab, the proposed studies are relevant to the German strain that caused the largest HUS epidemic in the world.